Manufacture of arrays with reduced error impact

ABSTRACT

Methods and apparatus are disclosed for synthesizing a plurality of compounds such as biopolymers on the surface of supports. The synthesis comprises a plurality of steps in which reagents for conducting the synthesis are deposited on the surface of the support to form precursors of the chemical compounds and, ultimately, the chemical compounds themselves. An error in the deposition may occur in one or more of the plurality of steps. A reagent for forming the chemical compounds is deposited on the surface of the support. A determination is made as to whether an error occurred in the depositing of the reagent. If an error is detected, the support is treated to re-deposit at least some of those reagents that were not correctly deposited. In one approach the support is treated to stabilize precursors of the chemical compounds, the source of the error is corrected, and the reagent applied above is re-deposited on the surface.

BACKGROUND OF THE INVENTION

This invention relates to the manufacturing of supports having bound tothe surfaces thereof a plurality of chemical compounds such as polymers,which are prepared on the surface in a series of steps. Moreparticularly, the present invention relates to methods for solid phasechemical synthesis, particularly solid phase synthesis of oligomerarrays, or attachment of oligonucleotides and polynucleotides tosurfaces, e.g., arrays of polynucleotides, where reagents are deliveredas droplets to the surface of a support.

In the field of diagnostics and therapeutics, it is often useful toattach species to a surface. One important application is in solid phasechemical synthesis wherein initial derivatization of a substrate surfaceenables synthesis of polymers such as oligonucleotides and peptides onthe substrate itself. Support bound oligomer arrays, particularlyoligonucleotide arrays and polypeptide arrays, may be used in screeningstudies for determination of binding affinity. Modification of surfacesfor use in chemical synthesis has been described. See, for example, U.S.Pat. No. 5,624,711 (Sundberg), U.S. Pat. No. 5,266,222 (Willis) and U.S.Pat. No. 5,137,765 (Farnsworth).

Determining the nucleotide sequences and expression levels of nucleicacids (DNA and RNA) is critical to understanding the function andcontrol of genes and their relationship, for example, to diseasediscovery and disease management. Analysis of genetic information playsa crucial role in biological experimentation. This has become especiallytrue with regard to studies directed at understanding the fundamentalgenetic and environmental factors associated with disease and theeffects of potential therapeutic agents on the cell. Such adetermination permits the early detection of infectious organisms suchas bacteria, viruses, etc.; genetic diseases such as sickle cell anemia;and various cancers. This paradigm shift has lead to an increasing needwithin the life science industries for more sensitive, more accurate andhigher-throughput technologies for performing analysis on geneticmaterial obtained from a variety of biological sources.

Unique or misexpressed nucleotide sequences in a polynucleotide can bedetected by hybridization with a nucleotide multimer, oroligonucleotide, probe. Hybridization is based on complementary basepairing. When complementary single stranded nucleic acids are incubatedtogether, the complementary base sequences pair to form double strandedhybrid molecules. These techniques rely upon the inherent ability ofnucleic acids to form duplexes via hydrogen bonding according toWatson-Crick base-pairing rules. The ability of single strandeddeoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) to form ahydrogen bonded structure with a complementary nucleic acid sequence hasbeen employed as an analytical tool in molecular biology research. Anoligonucleotide probe employed in the detection is selected with anucleotide sequence complementary, usually exactly complementary, to thenucleotide sequence in the target nucleic acid. Following hybridizationof the probe with the target nucleic acid, any oligonucleotideprobe/nucleic acid hybrids that have formed are typically separated fromunhybridized probe. The amount of oligonucleotide probe in either of thetwo separated media is then tested to provide a qualitative orquantitative measurement of the amount of target nucleic acid originallypresent.

Direct detection of labeled target nucleic acid hybridized tosurface-bound polynucleotide probes is particularly advantageous if thesurface contains a mosaic of different probes that are individuallylocalized to discrete, known areas of the surface. Such ordered arrayscontaining a large number of oligonucleotide probes have been developedas tools for high throughput analyses of genotype and gene expression.Oligonucleotides synthesized on a solid support recognize uniquelycomplementary nucleic acids by hybridization, and arrays can be designedto define specific target sequences, analyze gene expression patterns oridentify specific allelic variations. The arrays may be used forconducting cell study, for diagnosing disease, identifying geneexpression, monitoring drug response, determination of viral load,identifying genetic polymorphisms, analyze gene expression patterns oridentify specific allelic variations, and the like.

In one approach, cell matter is lysed, to release its DNA as fragments,which are then separated out by electrophoresis or other means, and thentagged with a fluorescent or other label. The resulting DNA mix isexposed to an array of oligonucleotide probes, whereupon selectivebinding to matching probe sites takes place. The array is then washedand interrogated to determine the extent of hybridization reactions. Inone approach the array is imaged so as to reveal for analysis andinterpretation the sites where binding has occurred. Arrays of differentchemical probe species provide methods of highly parallel detection, andhence improved speed and efficiency, in assays. Assuming that thedifferent sequence polynucleotides were correctly deposited inaccordance with the predetermined configuration, then the observedbinding pattern will be indicative of the presence and/or concentrationof one or more polynucleotide components of the sample.

Biopolymer arrays can be fabricated using either in situ synthesismethods or deposition of the previously obtained biopolymers. The insitu synthesis methods include those described in U.S. Pat. No.5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 andthe references cited therein for synthesizing polynucleotides(specifically, DNA). Such in situ synthesis methods can be basicallyregarded as iterating the sequence of depositing droplets of: (a) aprotected monomer onto predetermined locations on the surface of asupport to link with either a suitably activated surface or with apreviously deposited deprotected monomer; (b) deprotecting the depositedmonomer so that it can now react with a subsequently deposited protectedmonomer; and (c) depositing another protected monomer for linking.Different monomers may be deposited at different regions on thesubstrate during any one iteration so that the different regions of thecompleted array will have different desired biopolymer sequences. One ormore intermediate steps may be required in each iteration such as, forexample, capping or blocking, oxidation, deprotection of protectiongroups or deblocking, and washing steps.

In the deposition methods biopolymers are deposited at predeterminedlocations on a support surface that is suitably activated such that thebiopolymers can become linked to the surface. Biopolymers of differentsequence may be deposited at different regions of the substrate to yieldthe completed array. Washing or other additional steps may also be used.

Typical procedures are known in the art for deposition ofpolynucleotides, particularly DNA such as whole oligomers or cDNA. Onesuch procedure involves loading a small volume of DNA in solution in oneor more drop dispensers such as the tip of a pin or in an open capillaryand touching the pin or capillary to the surface of the substrate. Sucha procedure is described in U.S. Pat. No. 5,807,522. When the fluidtouches the surface, some of the fluid is transferred. The pin orcapillary must be washed prior to picking up the next type of DNA forspotting onto the array. This process is repeated for many differentsequences and, eventually, the desired array is formed.

In another approach reagents for in situ synthesis or DNA can be loadedinto a drop dispenser in the form of an inkjet head and fired onto thesurface of the support. Such a technique has been described, forexample, in PCT publications WO 95/25116 and WO 98/41531, and elsewhere.This method has the advantage of non-contact deposition. Other methodsinvolve pipetting apparatus and positive displacement pumps such as, forexample, the Biodot equipment available from Bio-Dot Inc., IrvineCalif., USA.

In array fabrication, there may be instances where an error occursduring the deposition of reagents on the surface of a support. Sucherrors may result, for example, from non-delivery of reagent from one ormore of dispensing elements such as the dispensing nozzles of an inkjetapparatus. Errors of this kind basically renders the support non-usablebecause one or more of the biopolymers deposited on the surface areincorrect. In instances of error occurrence, the particular synthesis isstopped, the support is discarded, the source of the error, e.g.,clogged printing nozzle, is fixed and a new synthesis is carried outwith a new support starting from the beginning of the syntheticprocedure. As may be appreciated, the occurrence of an error gives riseto a considerable amount of lost time, material and reagents.

There is a need, therefore, for a method for fabricating arrays suchthat errors that occur during the deposition of reagents on a supportare detected; the source of the error is repaired; the error itself iscorrected; and the fabrication is resumed with the same support.

SUMMARY OF THE INVENTION

One embodiment of the present invention is a method for synthesizing anarray of chemical compounds on the surface of a support. The synthesiscomprises a plurality of steps in which reagents for conducting thesynthesis are deposited on the surface of the support to form precursorsof the chemical compounds. One or more of the plurality of steps maycomprise an error in the deposition. Reagents for forming the chemicalcompounds are deposited on multiple locations on the surface. The abovestep is repeated in one or more cycles so as to form the chemicalcompounds. The reagents deposited in different cycles at the samelocations on the surface may or may not be the same. The methodadditionally comprises in at least one selected cycle determiningwhether an error occurred in the depositing of the first reagents in theselected cycle. If an error occurred, the source of the error iscorrected and at least some of those of the reagents that were notcorrectly deposited in the selected cycle are re-deposited on thesurface. Optionally, the support may be treated to stabilize precursorsof the chemical compounds after an error is detected.

Another embodiment of the present invention is a method for synthesizingan array of biopolymers on the surface of a support. A plurality ofsteps is employed in the synthesis wherein reagents comprisingbiopolymer subunits are deposited on multiple locations on the surfaceof the support. During the deposition an error may occur in one or moreof the plurality of steps. A support having a functionalized surface isplaced into a reaction chamber. A plurality of drops of a reagentcomprising a biopolymer subunit is dispensed to the surface from aplurality of nozzles. The above step is repeated in one or more cyclesso as to form the biopolymers. A determination is made as to whether anerror occurred in the dispensing of the reagent. If an error isdetected, the support is removed from the reaction chamber and placed ina holding chamber; the surface of the support in the holding chamber isstabilized; the source of the error is corrected; the support isreturned to the reaction chamber; and a plurality of drops of the abovereagents is dispensed to the surface from a plurality of nozzles. Thenormal steps of the synthesis are resumed where again a plurality ofdrops of a reagent comprising a biopolymer unit is dispensed to thesurface from a plurality of nozzles. The above steps are repeated toform the biopolymers of predetemmined characteristics.

Another embodiment of the present invention is an apparatus forsynthesizing an array of biopolymers on the surface of a support. Theapparatus comprises a reaction chamber, a mechanism for moving a supportto and from the reaction chamber, a controller for controlling themovement of the aforementioned mechanism, one or more fluid dispensingstations in fluid communication with the reaction chamber, a secondmechanism for determining the correct operation of the fluid dispensingstations, and a controller for controlling the second mechanism. Themechanism for determining the correct operation of the fluid dispensingstations is in communication with the controller for controlling themovement of the mechanism for moving a support to and from the reactionchamber. The apparatus further comprises a stabilization chamber forsubjecting the support to stabilization reagents. The apparatus mayoptionally comprise one or more additional chambers for conductingreactions that form part of the synthesis.

Another embodiment of the present invention is a method for synthesizinga plurality of chemical compounds on the surface of a support. Thesynthesis comprises a plurality of steps in which reagents forconducting the synthesis are deposited on the surface of the support toform precursors of the chemical compounds and, ultimately, the chemicalcompounds themselves. An error in the deposition may occur in one ormore of the plurality of steps. In the method in accordance with thepresent invention, a reagent for forming the chemical compounds isdeposited on the surface of the support. A determination is made as towhether an error occurred in the depositing of the reagent. If an erroris detected, then, in accordance with the present invention, the supportis treated to re-deposit at least those reagents that were not correctlydeposited because of the error in the deposition step. In one approachthe support is treated to stabilize precursors of the chemicalcompounds, the source of the error is corrected, and the reagent appliedin the step in which the error was detected is re-deposited on thesurface. In an alternative approach, if an error is detected, the erroris corrected and the reagents that were not correctly delivered to thesurface of the support are dispensed to the surface only at thelocations where reagent deposition did not occur because of the error.Subsequent to the aforementioned corrective approaches, the synthesis iscontinued in its normal pattern wherein a next a reagent for forming thechemical compounds is deposited on the surface. The above steps arerepeated to form the chemical compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart depicting one embodiment of a method inaccordance with the present invention.

FIG. 2 is a flow chart depicting an alternative embodiment of a methodin accordance with the present invention.

FIG. 3 is a schematic diagram depicting an embodiment of an apparatus inaccordance with the present invention.

FIG. 4 is a depiction of a surface of a support having numerous featuresprinted thereon and containing a missing feature due to a printingerror.

DETAILED DESCRIPTION OF THE INVENTION

The present methods and apparatus may be employed in the synthesis of aplurality of chemical compounds on supports with particular applicationto such synthesis on a commercial scale. The invention has applicationto the deposition of reagents for yielding the chemical compounds. Suchreagents may be, for example, chemical components for forming thechemical compounds, fully formed chemical compounds that are depositedon a surface, and so forth. Usually, the chemical compounds are thosethat are synthesized in a series of steps such as, for example, theaddition of building blocks, which are chemical components of thechemical compound. Examples of such building blocks include those foundin the synthesis of polymers such as, for example, subunits of thepolymers.

As mentioned above, the chemical compounds are those that aresynthesized in a series of steps, which usually involve linking togetherbuilding blocks that form the chemical compound. The invention hasparticular application to the synthesis of oligomers or polymers. Theoligomer or polymer is a chemical entity that contains a plurality ofmonomers. It is generally accepted that the term “oligomers” is used torefer to a species of polymers. The terms “oligomer” and “polymer” maybe used interchangeably herein. Polymers usually comprise at least twomonomers. Oligomers generally comprise about 6 to about 20,000 monomers,preferably, about 10 to about 10,000, more preferably about 15 to about4,000 monomers. Examples of polymers include polydeoxyribonucleotides,polyribonucleotides, other polynucleotides that are C-glycosides of apurine or pyrimidine base, or other modified polynucleotides,polypeptides, polysaccharides, and other chemical entities that containrepeating units of like chemical structure. Exemplary of oligomers areoligonucleotides and peptides.

A monomer is a chemical entity that can be covalently linked to one ormore other such entities to form an oligomer or polymer. Examples ofmonomers include nucleotides, amino acids, saccharides, peptoids, andthe like and subunits comprising nucleotides, amino acids, saccharides,peptoids and the like. The subunits may comprise all of the samecomponent such as, for example, all of the same nucleotide or aminoacid, or the subunit may comprise different components such as, forexample, different nucleotides or different amino acids. The subunitsmay comprise about 2 to about 2000, or about 5 to about 200, monomerunits. In general, the monomers have first and second sites (e.g.,C-termini and N-termini, or 5′ and 3′ sites) suitable for binding ofother like monomers by means of standard chemical reactions (e.g.,condensation, nucleophilic displacement of a leaving group, or thelike), and a diverse element that distinguishes a particular monomerfrom a different monomer of the same type (e.g., an amino acid sidechain, a nucleotide base, etc.). The initial substrate-bound, orsupport-bound, monomer is generally used as a building block in amulti-step synthesis procedure to form a complete ligand, such as in thesynthesis of oligonucleotides, oligopeptides, oligosaccharides, etc. andthe like.

A biomonomer references a single unit, which can be linked with the sameor other biomonomers to form a biopolymer (for example, a single aminoacid or nucleotide with two linking groups one or both of which may haveremovable protecting groups). A biomonomer fluid or biopolymer fluidreference a liquid containing either a biomonomer or biopolymer,respectively (typically in solution).

A biopolymer is a polymer of one or more types of repeating units.Biopolymers are typically found in biological systems and particularlyinclude polysaccharides (such as carbohydrates), and peptides (whichterm is used to include polypeptides, and proteins whether or notattached to a polysaccharide) and polynucleotides as well as theiranalogs such as those compounds composed of or containing amino acidanalogs or non-amino acid groups, or nucleotide analogs ornon-nucleotide groups. This includes polynucleotides in which theconventional backbone has been replaced with a non-naturally occurringor synthetic backbone, and nucleic acids (or synthetic or naturallyoccurring analogs) in which one or more of the conventional bases hasbeen replaced with a group (natural or synthetic) capable ofparticipating in Watson-Crick type hydrogen bonding interactions.

Polynucleotides are compounds or compositions that are polymericnucleotides or nucleic acid polymers. The polynucleotide may be anatural compound or a synthetic compound. Polynucleotides includeoligonucleotides and are comprised of natural nucleotides such asribonucleotides and deoxyribonucleotides and their derivatives althoughunnatural nucleotide mimetics such as 2′-modified nucleosides, peptidenucleic acids and oligomeric nucleoside phosphonates are also used. Thepolynucleotide can have from about 2 to 5,000,000 or more nucleotides.Usually, the oligonucleotides are at least about 2 nucleotides, usually,about 5 to about 100 nucleotides, more usually, about 10 to about 50nucleotides, and may be about 15 to about 30 nucleotides, in length.Polynucleotides include single or multiple stranded configurations,where one or more of the strands may or may not be completely alignedwith another.

A nucleotide refers to a sub-unit of a nucleic acid and has a phosphategroup, a 5 carbon sugar and a nitrogen containing base, as well asfunctional analogs (whether synthetic or naturally occurring) of suchsub-units which in the polymer form (as a polynucleotide) can hybridizewith naturally occurring polynucleotides in a sequence specific manneranalogous to that of two naturally occurring polynucleotides. Forexample, a “biopolymer” includes DNA (including cDNA), RNA,oligonucleotides, and PNA and other polynucleotides as described in U.S.Pat. No. 5,948,902 and references cited therein (all of which areincorporated herein by reference), regardless of the source. An“oligonucleotide” generally refers to a nucleotide multimer of about 10to 100 nucleotides in length, while a “polynucleotide” includes anucleotide multimer having any number of nucleotides.

The support to which a plurality of chemical compounds is attached isusually a porous or non-porous water insoluble material. The support canhave any one of a number of shapes, such as strip, plate, disk, rod,particle, and the like. The support can be hydrophilic or capable ofbeing rendered hydrophilic or it may be hydrophobic. The support isusually glass such as flat glass whose surface has been chemicallyactivated to support binding or synthesis thereon, glass available asBioglass and the like. However, the support may be made from materialssuch as inorganic powders, e.g., silica, magnesium sulfate, and alumina;natural polymeric materials, particularly cellulosic materials andmaterials derived from cellulose, such as fiber containing papers, e.g.,filter paper, chromatographic paper, etc.; synthetic or modifiednaturally occurring polymers, such as nitrocellulose, cellulose acetate,poly (vinyl chloride), polyacrylamide, cross linked dextran, agarose,polyacrylate, polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon,poly(vinyl butyrate), etc.; either used by themselves or in conjunctionwith other materials; ceramics, metals, and the like. Preferably, forpackaged arrays the support is a non-porous material such as glass,plastic, metal and the like.

The surface of a support is normally treated to create a primed orfunctionalized surface, that is, a surface that is able to support thesynthetic steps involved in the production of the chemical compound.Functionalization relates to modification of the surface of a support toprovide a plurality of functional groups on the support surface. By theterm “functionalized surface” is meant a support surface that has beenmodified so that a plurality of functional groups are present thereon.The manner of treatment is dependent on the nature of the chemicalcompound to be synthesized and on the nature of the support surface. Inone approach a reactive hydrophilic site or reactive hydrophilic groupis introduced onto the surface of the support. Such hydrophilic moietiescan be used as the starting point in a synthetic organic process.

In one embodiment, the surface of the support, such as a glass support,is siliceous, i.e., comprises silicon oxide groups, either present inthe natural state, e.g., glass, silica, silicon with an oxide layer,etc., or introduced by techniques well known in the art. One techniquefor introducing siloxyl groups onto the surface involves reactivehydrophilic moieties on the surface. These moieties are typicallyepoxide groups, carboxyl groups, thiol groups, and/or substituted orunsubstituted amino groups as well as a functionality that may be usedto introduce such a group such as, for example, an olefin that may beconverted to a hydroxyl group by means well known in the art. Oneapproach is disclosed in U.S. Pat. No. 5,474,796 (Brennan), the relevantportions of which are incorporated herein by reference. A siliceoussurface may be used to form silyl linkages, i.e., linkages that involvesilicon atoms. Usually, the silyl linkage involves a silicon-oxygenbond, a silicon-halogen bond, a silicon-nitrogen bond, or asilicon-carbon bond.

Another method for attachment is described in U.S. Pat. No. 6,219,674(Fulcrand, et al.). A surface is employed that comprises a linking groupconsisting of a first portion comprising a hydrocarbon chain, optionallysubstituted, and a second portion comprising an alkylene oxide or analkylene imine wherein the alkylene is optionally substituted. One endof the first portion is attached to the surface and one end of thesecond portion is attached to the other end of the first portion chainby means of an amine or an oxy functionality. The second portionterminates in an amine or a hydroxy functionality. The surface isreacted with the substance to be immobilized under conditions forattachment of the substance to the surface by means of the linkinggroup.

Another method for attachment is described in U.S. Pat. No. 6,258,454(Lefkowitz, et al.). A solid support having hydrophilic moieties on itssurface is treated with a derivatizing composition containing a mixtureof silanes. A first silane provides the desired reduction in surfaceenergy, while the second silane enables functionalization with molecularmoieties of interest, such as small molecules, initial monomers to beused in the solid phase synthesis of oligomers, or intact oligomers.Molecular moieties of interest may be attached through cleavable sites.

A procedure for the derivatization of a metal oxide surface uses anaminoalkyl silane derivative, e.g., trialkoxy 3-aminopropylsilane suchas aminopropyltriethoxy silane (APS), 4-aminobutyltrimethoxysilane,4-aminobutyltriethoxysilane, 2-aminoethyltriethoxysilane, and the like.APS reacts readily with the oxide and/or siloxyl groups on metal andsilicon surfaces. APS provides primary amine groups that may be used tocarry out the present methods. Such a derivatization procedure isdescribed in EP 0 173 356 B1, the relevant portions of which areincorporated herein by reference. Other methods for treating the surfaceof a support will be suggested to those skilled in the art in view ofthe teaching herein. Such methods include, for example, creatinghydroxyl terminated surfaces and so forth.

The apparatus and methods of the present invention are particularlyuseful in the synthesis of arrays of biopolymers. A biopolymer is apolymer of one or more types of repeating units relating to biology.Biopolymers are typically found in biological systems (although they maybe made synthetically) and particularly include polysaccharides such ascarbohydrates and the like, poly(amino acids) such as peptides includingpolypeptides and proteins, and polynucleotides, as well as suchcompounds composed of or containing amino acid analogs or non-amino acidgroups, or nucleotide analogs or non-nucleotide groups. This includespolynucleotides in which the conventional backbone has been replacedwith a non-naturally occurring or synthetic backbone, and nucleic acids(or synthetic or naturally occurring analogs) in which one or more ofthe conventional bases has been replaced with a group (natural orsynthetic) capable of participating in Watson-Crick type hydrogenbonding interactions.

An array includes any one, two or three dimensional arrangement ofaddressable regions bearing a particular chemical moiety or moietiessuch as, for example, biopolymers, e.g., one or more polynucleotides,associated with that region. An array is addressable in that it hasmultiple regions of different moieties, for example, differentpolynucleotide sequences, such that a region or feature or spot of thearray at a particular predetermined location or address on the array candetect a particular target molecule or class of target moleculesalthough a feature may incidentally detect non-target molecules of thatfeature. Where a predetermined arrangement of arrays is desired, any ofa variety of geometries may be constructed. In one approach the arraymay be in the form of organized rows and columns of features. Inalternative approaches, arrays can be arranged in a series ofcurvilinear rows across the surface of a support such as, for example, aseries of concentric circles or semi-circles of spots, and the like.

The present methods and apparatus may be used in the synthesis ofpolypeptides. The synthesis of polypeptides involves the sequentialaddition of amino acids to a growing peptide chain. This approachcomprises attaching an amino acid to the functionalized surface of thesupport. In one approach the synthesis involves sequential addition ofcarboxyl-protected amino acids to a growing peptide chain with eachadditional amino acid in the sequence similarly protected and coupled tothe terminal amino acid of the oligopeptide under conditions suitablefor forming an amide linkage. Such conditions are well known to theskilled artisan. See, for example, Merrifield, B. (1986), Solid PhaseSynthesis, Sciences 232, 341-347. After polypeptide synthesis iscomplete, acid is used to remove the remaining terminal protectinggroups.

The present invention has particular application to the synthesis ofarrays of chemical compounds on a surface of a support. Typically,methods and apparatus of the present invention generate or use an arrayassembly that may include a support carrying one or more arrays disposedalong a surface of the support and separated by inter-array areas.Normally, the surface of the support opposite the surface with thearrays does not carry any arrays. The arrays can be designed for testingagainst any type of sample, whether a trial sample, a reference sample,a combination of the foregoing, or a known mixture of components such aspolynucleotides, proteins, polysaccharides and the like (in which casethe arrays may be composed of features carrying unknown sequences to beevaluated). The surface of the support may carry at least one, two,four, or at least ten, arrays. Depending upon intended use, any or allof the arrays may be the same or different from one another and each maycontain multiple spots or features of chemical compounds such as, e.g.,biopolymers in the form of polynucleotides or other biopolymer. Atypical array may contain more than ten, more than one hundred, morethan one thousand or ten thousand features, or even more than onehundred thousand features, in an area of less than 20 cm² or even lessthan 10 cm². For example, features may have widths (that is, diameter,for a round spot) in the range from a 10 μm to 1.0 cm. In otherembodiments each feature may have a width in the range of 1.0 μm to 1.0mm, usually 5.0 μm to 500 μm, and more usually 10 μm to 200 μm.Non-round features may have area ranges equivalent to that of circularfeatures with the foregoing width (diameter) ranges. At least some, orall, of the features are of different compositions (for example, whenany repeats of each feature composition are excluded, the remainingfeatures may account for at least 5%, 10%, or 20% of the total number offeatures).

Each feature, or element, within the molecular array is defined to be asmall, regularly shaped region of the surface of the substrate. Thefeatures are arranged in a predetermined manner. Each feature of anarray usually carries a predetermined chemical compound or mixturesthereof. Each feature within the molecular array may contain a differentmolecular species, and the molecular species within a given feature maydiffer from the molecular species within the remaining features of themolecular array. Some or all of the features may be of differentcompositions. Each array may contain multiple spots or features and eacharray may be separated by spaces or areas. It will also be appreciatedthat there need not be any space separating arrays from one another.Interarray areas and interfeature areas are usually present but are notessential. These areas do not carry any chemical compound such aspolynucleotide (or other biopolymer of a type of which the features arecomposed). Interarray areas and interfeature areas typically will bepresent where arrays are formed by the conventional in situ process orby deposition of previously obtained moieties, as described above, bydepositing for each feature at least one droplet of reagent such as froma pulse jet (for example, an inkjet type head) but may not be presentwhen, for example, photolithographic array fabrication processes areused. It will be appreciated though, that the interarray areas andinterfeature areas, when present, could be of various sizes andconfigurations.

Each array may cover an area of less than 100 cm², or even less than 50cm², 10 cm² or 1 cm². In many embodiments, the support (sometimesreferenced as a “substrate”) carrying the one or more arrays may beshaped generally as a rectangular solid (although other shapes arepossible), having a length of more than 4 mm and less than 1 m, usuallymore than 4 mm and less than 600 mm, more usually less than 400 mm; awidth of more than 4 mm and less than 1 m, usually less than 500 mm andmore usually less than 400 mm; and a thickness of more than 0.01 mm andless than 5.0 mm, usually more than 0.1 mm and less than 2 mm and moreusually more than 0.2 and less than 1 mm. With arrays that are read bydetecting fluorescence, the substrate may be of a material that emitslow fluorescence upon illumination with the excitation light.Additionally, in this situation the substrate may be relativelytransparent to reduce the absorption of the incident illuminating laserlight and subsequent heating if the focused laser beam travels tooslowly over a region. For example, the substrate may transmit at least20%, or 50% (or even at least 70%, 90%, or 95%), of the illuminatinglight incident on the front as may be measured across the entireintegrated spectrum of such illuminating light or alternatively at 532nm or 633 nm. Flexible or rigid substrates may be used.

The devices and methods of the present invention are particularly usefulin the synthesis of oligonucleotide arrays for determinations ofpolynucleotides. As explained briefly above, in the field of bioscience,arrays of oligonucleotide probes, fabricated or deposited on a surfaceof a support, are used to identify DNA sequences in cell matter. Thearrays generally involve a surface containing a mosaic of differentoligonucleotides or sample nucleic acid sequences or polynucleotidesthat are individually localized to discrete, known areas of the surface.In one approach, multiple identical arrays across a complete frontsurface of a single substrate or support are used.

Ordered arrays containing a large number of oligonucleotides have beendeveloped as tools for high throughput analyses of genotype and geneexpression. Oligonucleotides synthesized on a solid support recognizeuniquely complementary nucleic acids by hybridization, and arrays can bedesigned to define specific target sequences, analyze gene expressionpatterns or identify specific allelic variations. The arrays may be usedfor conducting cell study, for diagnosing disease, identifying geneexpression, monitoring drug response, determination of viral load,identifying genetic polymorphisms, analyze gene expression patterns oridentify specific allelic variations, and the like.

The synthesis of arrays of polynucleotides on the surface of a supportusually involves attaching an initial nucleoside or nucleotide to afunctionalized surface. The surface may be functionalized as discussedabove. In one approach the surface is reacted with nucleosides ornucleotides that are also functionalized for reaction with the groups onthe surface of the support. Methods for introducing appropriate aminespecific or alcohol specific reactive functional groups into anucleoside or nucleotide include, by way of example, addition of aspacer amine containing phosphoramidites, addition on the base ofalkynes or alkenes using palladium mediated coupling, addition of spaceramine containing activated carbonyl esters, addition of boronconjugates, formation of Schiff bases.

After the introduction of the nucleoside or nucleotide onto the surface,the attached nucleotide may be used to construct the polynucleotide bymeans well known in the art. For example, in the synthesis of arrays ofoligonucleotides, nucleoside monomers are generally employed. In thisembodiment an array of the above compounds is attached to the surfaceand each compound is reacted to attach a nucleoside. Nucleoside monomersare used to form the polynucleotides usually by phosphate coupling,either direct phosphate coupling or coupling using a phosphate precursorsuch as a phosphite coupling. Such coupling thus includes the use ofamidite (phosphoramidite), phosphodiester, phosphotriester,H-phosphonate, phosphite halide, and the like coupling.

One preferred coupling method is phosphoramidite coupling, which is aphosphite coupling. In using this coupling method, after the phosphitecoupling is complete, the resulting phosphite is oxidized to aphosphate. Oxidation can be effected with iodine to give phosphates orwith sulfur to give phosphorothioates. The phosphoramidites aredissolved in anhydrous acetonitrile to give a solution having a givenratio of amidite concentrations. The mixture of known chemicallycompatible monomers is reacted to a solid support, or further along, maybe reacted to a growing chain of monomer units, which may also bereferred to as the polymer precursors. In one particular example, theterminal 5′-hydroxyl group is caused to react with adeoxyribonucleoside-3′-O-(N,N-diisopropylamino)phosphor-amiditeprotected at the 5′-position with dimethoxytrityl or the like. The 5′protecting group is removed after the coupling reaction, and theprocedure is repeated with additional protected nucleotides untilsynthesis of the desired polynucleotide is complete. For a more detaileddiscussion of the chemistry involved in the above synthetic approaches,see, for example, U.S. Pat. No. 5,436,327 at column 2, line 34, tocolumn 4, line 36, which is incorporated herein by reference in itsentirety.

Various ways may be employed to introduce the reagents for producing anarray of polynucleotides on the surface of a support such as a glasssupport. Such methods are known in the art and include methods involvingdispensing reagents to the surface of a support in the form of droplets.One in situ method employs inkjet printing technology to dispense theappropriate phosphoramidite reagents and other reagents onto individualsites on a surface of a support. Oligonucleotides are synthesized on asurface of a substrate in situ using phosphoramidite chemistry.Solutions containing nucleotide monomers and other reagents as necessarysuch as an activator, e.g., tetrazole, are applied to the surface of asupport by means of ink-jet technology such as, e.g., thermal ink-jettechnology or piezo inkjet technology. Individual droplets of reagentsare applied to reactive areas on the surface using, for example, athermal ink-jet type nozzle or piezo inkjet technology. The surface ofthe support may have a double bond terminated alkyl trichlorosilanecoating, which is reacted in a hydroboration reaction to provideterminal hydroxyl groups. These hydroxyl groups provide for linking to aterminal primary amine group on a monomeric reagent. Excess ofnon-reacted chemical on the surface is washed away in a subsequent step.For example, see U.S. Pat. No. 5,700,637 and PCT WO 95/25116 and PCTapplication WO 89/10977.

For in situ fabrication methods, multiple different reagent droplets aredeposited on the surface of a support at a given target location inorder to form the final feature (hence a probe of the feature issynthesized on the array substrate). Deposition may be by, for example,pulsejet or other similar means. The in situ fabrication methods includethose described in U.S. Pat. No. 5,449,754 for synthesizing peptidearrays, and in U.S. Pat. No. 6,180,351 and WO 98/41531 and thereferences cited therein for polynucleotides, and may also use pulsejetsfor depositing reagents. The in situ method for fabricating apolynucleotide array typically follows, at each of the multipledifferent addresses at which features are to be formed, the sameconventional iterative sequence used in forming polynucleotides fromnucleoside reagents on a support by means of known chemistry.

This iterative sequence can be considered as multiple ones of thefollowing attachment cycle at each feature to be formed: (a) coupling anactivated selected nucleoside (a monomeric unit) through a phosphitelinkage to a functionalized support in the first iteration, or anucleoside bound to the substrate (i.e. the nucleoside-modifiedsubstrate) in subsequent iterations; (b) optionally, blocking unreactedhydroxyl groups on the substrate bound nucleoside (sometimes referencedas “capping”); (c) oxidizing the phosphite linkage of step (a) to form aphosphate linkage; and (d) removing the protecting group(“deprotection”) from the now substrate bound nucleoside coupled in step(a), to generate a reactive site for the next cycle of these steps. Thecoupling can be performed by depositing drops of an activator andphosphoramidite at the specific desired feature locations for the array.Capping, oxidation and deprotection can be accomplished by treating theentire substrate (“flooding”) with a layer of the appropriate reagent.The functionalized support (in the first cycle) or deprotected couplednucleoside (in subsequent cycles) provides a substrate bound moiety witha linking group for forming the phosphite linkage with a next nucleosideto be coupled in step (a). Final deprotection of nucleoside bases can beaccomplished using alkaline conditions such as ammonium hydroxide, inanother flooding procedure in a known manner. Conventionally, a singlepulsejet or other dispenser is assigned to deposit a single monomericunit.

Another approach for fabricating an array of biopolymers on a substrateusing a biopolymer or biomonomer fluid and using a fluid dispensing headis described in U.S. Pat. No. 6,242,266 (Schleifer, et al.). The headhas at least one jet that can dispense droplets onto a surface of asupport. The jet includes a chamber with an orifice and an ejector,which, when activated, causes a droplet to be ejected from the orifice.Multiple droplets of the biopolymer or biomonomer fluid are dispensedfrom the head orifice so as to form an array of droplets on the surfaceof the substrate.

In another embodiment (U.S. Pat. No. 6,232,072) (Fisher) a method of,and apparatus for, fabricating a biopolymer array is disclosed. Dropletsof fluid carrying the biopolymer or biomonomer are deposited onto afront side of a transparent substrate. Light is directed through thesubstrate from the front side, back through a substrate back side and afirst set of deposited droplets on the first side to an image sensor.

An example of another method for chemical array fabrication is describedin U.S. Pat. No. 6,180,351 (Cattell). The method includes receiving froma remote station information on a layout of the array and an associatedfirst identifier. A local identifier is generated corresponding to thefirst identifier and associated array. The local identifier is shorterin length than the corresponding first identifier. The addressable arrayis fabricated on the substrate in accordance with the received layoutinformation.

The foregoing chemistry of the synthesis of polynucleotides is describedin detail, for example, in Caruthers, Science 230: 281-285, 1985;Itakura, et al., Ann. Rev. Biochem. 53: 323-356; Hunkapillar, et al.,Nature 310: 105-110, 1984; and in “Synthesis of OligonucleotideDerivatives in Design and Targeted Reaction of OligonucleotideDerivatives”, CRC Press, Boca Raton, Fla., pages 100 et seq., U.S. Pat.Nos. 4,458,066, 4,500,707, 5,153,319, 5,869,643 and European patentapplication, EP 0294196, and elsewhere. The phosphoramidite andphosphite triester approaches are most broadly used, but otherapproaches include the phosphodiester approach, the phosphotriesterapproach and the H-phosphonate approach. The substrates are typicallyfunctionalized to bond to the first deposited monomer. Suitabletechniques for functionalizing substrates with such linking moieties aredescribed, for example, in Southern, E. M., Maskos, U. and Elder, J. K.,Genomics, 13, 1007-1017, 1992.

In the case of array fabrication, different monomers and activator maybe deposited at different addresses on the substrate during any onecycle so that the different features of the completed array will havedifferent desired biopolymer sequences. As explained above, one or moreintermediate steps may be required in each cycle, such as theconventional oxidation, capping or blocking, deprotection of protectinggroups or deblocking, and washing steps in the case of in situfabrication of polynucleotide arrays; again, these steps may beperformed in flooding procedure.

As is well known in the art of ink jet printing, the amount of fluidthat is expelled in a single activation event of a pulse jet can becontrolled by changing one or more of a number of parameters, includingthe orifice diameter, the orifice length (thickness of the orificemember at the orifice), the size of the deposition chamber, and the sizeof the heating element, among others. The amount of fluid that isexpelled during a single activation event is generally in the rangeabout 0.1 to about 1000 pL, usually about 0.5 to about 500 pL and moreusually about 1.0 to about 250 pL. A typical velocity at which the fluidis expelled from the chamber is more than about 1 m/s, usually more thanabout 10 m/s, and may be as great as about 20 m/s or greater. As will beappreciated, if the orifice is in motion with respect to the receivingsurface at the time an ejector is activated, the actual site ofdeposition of the material will not be the location that is at themoment of activation in a line-of-sight relation to the orifice, butwill be a location that is predictable for the given distances andvelocities.

The spots can have widths (such as, for example, diameter for a roundspot) in the range from a minimum of about 10 μm to a maximum of about1.0 cm. In embodiments where very small spot sizes or feature sizes aredesired, material can be deposited in small spots whose width is in therange about 1.0 μm to about 1.0 mm, usually about 5.0 μm to about 500μm, and more usually about 10 μm to 200 μm.

In the method in accordance with the present invention, a reagent forforming the chemical compounds, such as, for example, a nucleotidereagent or a polynucleotide reagent, is deposited on the surface of thesupport as discussed above. Usually, a deposition sequence is initiatedto deposit the desired fluid droplets containing nucleotide reagents onthe surface of a support to provide dried drops on the surface accordingto the predetermined arrangement of the target, each with respectivetarget locations and dimensions. In this sequence a processor causes apositioning system to position a head facing the surface of the supportat an appropriate distance from the surface. The processor then causesthe positioning system to scan the head across the surface line by line(or in some other desired pattern), while coordinating activation of theejectors in the head so as to dispense droplets in accordance with thetarget pattern. If necessary or desired, the processor can repeat theload and dispensing sequences one or more times until the head hasdispensed the desired number of droplets in accordance with theparticular reaction step of the array formation. The number of dropsdispensed in any one reaction step can be, for example, from about 1 toabout 10, usually, about 3 to about 5. The total number of spots on thesurface of the support may be, for example, at least about ten, at leastabout one hundred, at least about one thousand, or at least about onehundred thousand.

At the conclusion of the droplet dispensing for the reaction step, adetermination is made as to whether one or more errors occurred duringthe deposition step. If during the deposition sequence all droplets werecorrectly deposited, they would yield the predetermined arrangement oftarget polynucleotides on the surface of the support. In practice,however, an error may occur in the deposition, which would result in anincorrect array pattern. Prior to the present invention supports havingerrors of the sort described above were discarded.

The nature of the determination as to whether an error has occurred inthe deposition of the reagents depends on the nature of the error. Theerror may be a misfire of a nozzle, e.g., ink jet head, delivering areagent to a spot or multiplicity of spots on the surface of a support.Other errors include, for example, electronic error (e.g., inkjetcontroller), artifact on surface of support, build up of material oninkjet head, satellite formation from nozzle, etc. At any layerrepresented by the depositing of reagents in one step of a sequence ofsteps, two groups of features may be formed in the event of a misprintdue to an error. One group of features may have received necessaryreagents and the coupling of the reagent to the growing molecule atvarious features on the support is successfully carried out. Anothergroup of features may not have received necessary reagents and,therefore, any coupling reactions would be incomplete. If the subsequentsteps of the synthesis (such as in this example washing of the surface,oxidation, washing, deblocking and washing) are carried out, variousfeatures on the surface will be incorrect. If the subsequent steps ofthe synthesis also contain the optional capping step, various featureson the surface will be terminated and unusable in hybridizationexperiments.

In accordance with the present invention, a determination is made as towhether an error occurred in a particular cycle. The occurrence of anerror may be determined by the use of an inspection station having animaging system, which includes a camera to capture one or more images ofthe surface of a support on which the deposited droplets have dried toform spots. The camera is mounted for movement to facilitate imagecapture across the entire surface of the support although a suitablecamera could be located in a fixed position if desired. However, sincehigh resolution images are usually required from the camera, and since atypical substrate may be about 12 inches by 12 inches, the camera willnot likely be able to yield images of the required resolution of allarrays on a given support simultaneously. Thus, precision movement ofthe camera may be required. Of course, the light sensor of a cameracould potentially be mounted elsewhere, with a light-receiving element(such as a mirror) mounted for movement and arranged to direct light tothe sensor (using other moving and/or stationary mirrors, for example).Any suitable analog or digital image capture device (including a line byline scanner) can be used as the camera, although, if an analog camerais used, the processor should include a suitable analog/digitalconverter. In addition, more than one camera can be used if desired.

The support may have any desired dimension. However, the camera usuallyshould have sufficient resolution to permit it to distinguish andobserve each spot on the surface of the support. Movement of the camerawith a moving device such as, e.g., a head retainer, facilitates itscanning over the entire surface of the support and its capturing ofmultiple images with sufficient resolution such that a good image ofeach spot of each array is obtained. The camera should have a resolutionthat provides a pixel size of about 1 to about 100 micrometers and moretypically about 4 to about 10 micrometers.

In addition to imaging and analysis of every active feature afterprinting, a print test may be performed. During a print test, reagentsfrom all nozzles and from all print heads are printed in close proximityon a solid support. The test print area is consequently imaged andanalyzed for missing features. In the case where one or several featuresare missing, such as in the example shown in FIG. 4, the identity of themisfiring nozzle(s) may be inferred from the relative position of themissing feature on the support. Appropriate action on the malfunctioningnozzle(s) may be taken. The advantage of the test print over imaging ofall features is the rapidity by which malfunctioning nozzles arediagnosed.

The images from the camera are observed after each cycle in the reactionscheme to determine whether an error has occurred in the deposition ofthe reagents. If an error is detected, then, in accordance with thepresent invention, the printing process is stopped and the support istreated to stabilize precursors of the chemical compounds on itssurface. The nature of the stabilization of the precursors is dependenton the nature of the precursors. For example, the precursors may beaddition polymers of monomeric units such as nucleotides and thereaction scheme utilizes phosphoramidite chemistry. In accordance withthe reaction scheme, in any one cycle the precursors on the surface ofthe support are oxidized, as discussed above, to yield, e.g., aphosphate from a phosphite. In such a situation, the precursors thathave been successfully printed usually have a protecting group. On theother hand, the precursors in the spots where an error has occurredusually have no protecting group since the surface was primed forreaction with the reagents delivered in a particular cycle.

In accordance with one aspect of the present invention, when an error isidentified, the support is isolated from the printing chamber andtreated to stabilize the material on the surface of the support. In thephosphoramidite coupling method of preparing polynucleotides,stabilization of the surface of the support may be achieved by simplywashing the surface with, for example, acetonitrile. The primaryconsideration with respect to the stabilization of the materials on thesurface of the support is that the materials on the surface be protectedfrom any significant degradation, which might result in an incorrectarray arrangement. To provide further protection in the stabilizationstep in the phosphoramidite coupling method, the surface of the supportmay be subjected to an oxidation step, i.e., oxidizing the materials,under conditions that do not result in cleavage of any protecting groupspresent. The oxidizing step may be carried out prior to or after awashing step in a manner similar to the oxidation step in the syntheticscheme. In one exemplary approach, the surface of the support may besubjected to an oxidation step followed by two separate wash steps,usually, with acetonitrile although other suitable wash solutions asmentioned below may be employed depending on the nature of the oxidationstep. Alternatively, the surface of the support may be washed, subjectedto an oxidation step and then washed again. Other approaches may also beemployed. Following a wash step, whether or not employed in conjunctionwith an oxidation step, the surface of the support may be dried as isknown in the art. No capping or deprotection of protecting groups isnormally performed during the stabilization steps.

As indicated above, as part of the reaction scheme the precursors at thespots on the surface of the support have been deprotected as part of thesynthesis cycle. Thus, the precursors are reactive to the reagentsdeposited on the surface during the cycle in question. Accordingly,reagents successfully deposited at spots on the surface react with thematerial at the specific locations on the surface of the support and theresultant product has a protecting group present due to the presence ofa protecting group on the reagent that has been deposited. Thus, theresultant products at those spots where a successful deposition hasoccurred have a protecting group and an oxidation step does not affectsuch materials. On the other hand, spots where reagents were notsuccessfully deposited have materials that do not have a protectinggroup, which was removed prior to the particular deposition step.Accordingly, the oxidation step results in oxidation of material atthose spots where a reagent deposition step has failed.

Subsequent to the stabilization step, the support is then subjected to acycle in the reaction scheme, which is the same as the cycle in whichthe error in deposition occurred. In other words the reagent applied inthe cycle in which an error in deposition occurred is re-deposited onthe surface. Reagents deposited at spots where the materials have aprotecting group do not react with the material at those spots. Reagentsdeposited at spots that have materials with no protecting groups reactwith the materials at those spots. In this way the particular cycle inthe reaction scheme is completed even though an error had occurred, andthe remainder of the cycles in the reaction scheme may then be completedto yield the chemical compounds at the feature sites. In accordance withthe present invention, an examination is made during each cycle todetermine whether an error has occurred in the deposition step and theprocedure discussed above is repeated to compensate for such errors whendetected. Thus, after error detection and correction, the synthesis iscontinued in its normal pattern wherein subsequent reagents for formingthe chemical compounds are deposited on the surface. The above steps arerepeated until the chemical compounds are formed.

As mentioned above, the steps of capping, oxidation and deprotection canbe accomplished by treating the entire surface of a support with a layerof the appropriate reagent, which is often referred to as a floodingstep. Some or all of the above steps may be performed using flow cells.Accordingly, for example, after addition of a nucleoside monomer, suchas depositing the reagent using an ink jet method, the support is placedinto a chamber of a flow cell, which is typically a housing having areaction cavity or chamber disposed therein. The flow cell allows fluidsto be passed through the chamber where the support is disposed. Thesupport may be mounted in the chamber in or on a holder. The housingusually further comprises at least one fluid inlet and at least onefluid outlet for flowing fluids into and through the chamber in whichthe support is mounted. In one approach, the fluid outlet may be used tovent the interior of the reaction chamber for introduction and removalof fluid by means of the inlet. On the other hand, fluids may beintroduced into the reaction chamber by means of the inlet with theoutlet serving as a vent and fluids may be removed from the reactionchamber by means of the outlet with the inlet serving as a vent.

The inlet of the flow cell is usually in fluid communication with anelement that controls the flow of fluid into the flow cell such as, forexample, a manifold, a valve, and the like or combinations thereof. Thiselement in turn is in fluid communication with one or more fluid reagentdispensing stations. In this way different fluid reagents for one stepin the synthesis of the chemical compound may be introduced sequentiallyinto the flow cell. These reagents may be, for example, wash fluids,oxidizing agents, reducing agents, blocking or protecting agents,unblocking (deblocking) or deprotecting agents, and so forth. Anyreagent that is normally a solid reagent may be converted to a fluidreagent by dissolution in a suitable solvent, which may be a proticsolvent or an aprotic solvent. The solvent may be an organic solventsuch as, by way of illustration and not limitation, organic solvents offrom 1 to about 6, more usually from 1 to about 4, carbon atoms,including alcohols such as methanol, ethanol, propanol, etc., etherssuch as tetrahydrofuran, ethyl ether, propyl ether, etc., acetonitrile,dimethylfommamide, dimethylsulfoxide, and the like. In somecircumstances, the solvent may be an aqueous medium that is solely wateror may contain a buffer, or may contain from about 0.01 to about 80 ormore volume percent of a cosolvent such as an organic solvent asmentioned above.

The amount of the reagents employed in each synthetic step in the methodof the present invention is dependent on the nature of the reagents,solubility of the reagents, reactivity of the reagents, availability ofthe reagents, purity of the reagents, and so forth. Such amounts shouldbe readily apparent to those skilled in the art in view of thedisclosure herein. Usually, stoichiometric amounts are employed, butexcess of one reagent over the other may be used where circumstancesdictate. Typically, the amounts of the reagents are those necessary toachieve the overall synthesis of the chemical compound in accordancewith the present invention. The time period for conducting the presentmethod is dependent upon the specific reaction and reagents beingutilized and the chemical compound being synthesized.

Using as an example the synthesis of polynucleotides on a surface by thephosphoramidite method, the step of oxidation to stabilize the surfaceof the support may be carried out in a dedicated flow cell. Accordingly,following addition of a monomer and discovery of an error in deposition,the support may be placed in the flow cell. Various fluid dispensingstations are connected by means of a manifold and suitable valves to theinlet of the flow cell. Each of the fluid dispensing stations contains adifferent fluid reagent involved in performing the particular stepsinvolved in the stabilization procedure. Thus, in this example, onestation may contain an oxidizing agent for oxidizing the phosphite tothe phosphate and another station may contain a wash reagent such asacetonitrile.

As mentioned above, various approaches may be taken in carrying out thestep for stabilizing the materials on the surface of a support once anerror in deposition has been discovered. For example, the surface may besubjected to an oxidation step followed by consecutive wash steps.Accordingly, after the support has been isolated and moved to a flowcell, the oxidizing agent is allowed to pass into and out of the flowcell and the surface is then washed with the wash reagent as describedabove. After a drying step, the support is returned to the printingchamber where the surface is re-printed with the same reagent as in themis-printing step. The normal repetition of cycles in the reactionscheme is then resumed.

After the printing step in any one cycle where no error in depositionwas detected or where an error was detected and appropriate correctionwas made, the support may be removed from the printing chamber andsubjected to steps for preparing the support for the printing step in anext cycle, i.e., dispensing of reagent for synthesizing the biopolymer.The steps for preparing the support for the next printing step depend onthe method of synthesis. For example, where the phosphoramidite couplingmethod is employed, such steps include washing, optionally capping,oxidizing, deblocking or removal of a protecting group, and so forth. Tothis end in one approach the support is placed in a flow cell. Washreagent is first allowed to pass into and out of the flow cell. Next,oxidizing agent is introduced into the flow cell. The support is thensubjected to a deblocking step, which may be carried out in the sameflow cell or a different flow cell. Accordingly, the support may betransported from a first flow cell to a second flow cell. At this point,a deblocking reagent for removing a protecting group is allowed to passinto and out of the second flow cell. The deblocking reagent iscontained in a fluid dispensing station that is in fluid communicationwith the second flow cell. Next, wash reagent contained in a fluiddispensing station that is in fluid communication with the second flowcell is passed into and out of the second flow cell. Following the abovesynthetic steps, the support is transported from the second flow cell tothe printing chamber where the next monomer addition in a subsequentcycle is carried out and the above repetitive synthetic steps areconducted as discussed above.

The following discussion is by way of illustration and not limitation.Referring to FIG. 1 a print step is carried out to place reagents on thesurface of a support in predetermined locations. A determination is madeas to whether an error occurred as a result of the failure of one ormore of the printing elements involved in the printing process. If noerror occurred, the support is subjected to, optionally a capping step,an oxidation step and a deblocking step as is customarily employed inphosphoramidite coupling. The support is then returned to the printingchamber for the next print step in the reaction scheme. Theaforementioned sequence of steps is repeated. If the error determinationindicates that a failure in the printing process occurred, the supportis isolated and the surface is treated to stabilize the materials on thesurface. Following stabilization, the support is subjected to the sameprinting step in which the error occurred. In other words the cycle inwhich the error occurred is repeated, i.e., the support is subjected toa re-printing step. Following the re-printing step, error determinationis again carried out. Depending on the outcome of such determination,the appropriate sequence is followed as indicated in FIG. 1. As can beseen in FIG. 1, if an error is determined, the source of the error isrepaired prior to the re-printing process.

As an alternative approach to that discussed above, it is within thepurview of the present invention, to determine whether an error hasoccurred in any one cycle and then to reprint only those spots where anerror in deposition occurred. In this approach a program is employed toascertain which of the dispensing elements has caused an error. Theprogram then activates a re-print cycle where the correct reagents aredeposited in the locations where the failure to deposit reagentsoccurred. The support is then removed from the printing chamber andtreated to stabilize the surface of the support as discussed above. Thedispensing elements that caused the error are repaired. The reactionscheme is then resumed by beginning the next cycle in the addition ofthe monomer units.

The aforementioned alternative approach is depicted as a flow chart inFIG. 2. Referring to FIG. 2 a print step is carried out to placereagents on the surface of a support in predetermined locations. Adetermination is made as to the occurrence and location of an error as aresult of the failure of one or more of the printing elements involvedin the printing process. If no error occurred, the support is subjectedto, optionally a capping step, an oxidation step and a deblocking stepas is customarily employed in phosphoramidite coupling. The support isthen returned to the printing chamber for the next print step in thereaction scheme. The aforementioned sequence of steps is repeated. Ifthe error determination indicates that a failure in the printing processoccurred, the support is retained in the printing chamber and thesupport is subjected to the same printing step in which the erroroccurred where only the specific locations that were affected arere-printed. In other words the cycle in which the error occurred isrepeated only for those specific locations. Following the re-printingstep, error determination is again carried out. Depending on the outcomeof such determination, the appropriate sequence is followed as indicatedin FIG. 2. As can be seen in FIG. 2, if an error is determined, thesource of the error is repaired prior to the re-printing process.

In yet another approach a print step is carried out and the printedfeatures are then checked for errors. If errors are detected, allfeatures are reprinted, that is, the missing features plus the ones thatwere deposited correctly.

Various apparatus may be employed in carrying out the present invention.One such apparatus comprises a platform and a plurality of flow cellsmounted on the platform wherein one of the flow cells is dedicated tostabilization of supports on which errors have occurred in deposition ofreagents. The flow cells comprise a chamber, a holder for the support,at least one inlet and an outlet, wherein each of the inlets is in fluidcommunication with a manifold. One or more fluid dispensing stations aremounted on the platform and are in fluid communication with one or moreof the plurality of flow cells by means of the manifolds. A station formonomer addition to the surface of the support, for example, a stationcomprising one or more printing heads, is mounted on the platform. Theapparatus also comprises a mechanism for moving a support to and fromthe station for monomer addition and a flow cell and from one flow cellto another flow cell. The mechanism may be, for example, a robotic arm,and so forth. The mechanism for moving a support is in communicationsuch as, for example, electrical communication, with a mechanism fordetermining whether an error occurred in the deposition of reagents inthe station for monomer addition. As a result of such communication, themechanism for moving the support may be activated to move the supportfrom the reaction chamber to a stabilization chamber that is dedicatedto subjecting the support to stabilizing reagents.

In an alternative embodiment of an appropriate apparatus in accordancewith one aspect of the present invention, a dedicated stabilizationchamber is not employed. The apparatus comprises a mechanism foractivating only those dispensing elements, e.g., print heads, which weredetermined to have resulted in an error in the deposition of thereagents. Thus, once a determination of an error is made, the dispensingelements that caused the error in deposition are repaired or replaced.Next, reagents of the cycle in which an error occurred are dispensedonly to the sites on the support at which incorrect deposition wasdetected. Accordingly, the dispensing elements may be activated todispense only those reagents that were not dispensed or incorrectlydispensed to locations on the support in the step of the synthesis inquestion. Such a mechanism comprises, in one exemplary approach, loadingthe printing pattern for the step of the synthesis in question (reagentvs. position map), masking out the positions that were dispensedcorrectly, and re-printing the step using the masked pattern.

In one embodiment of a mechanism for moving a support from one flow cellto another flow cell or from the print chamber to a flow cell or astabilization flow cell, the support is delivered into the opening inthe wall of the flow cell housing by engagement with a holding element,which usually comprises a main arm and an end portion that contacts andengages a surface of the support. In one embodiment the holding elementis in the form of a fork that is vacuum activated. Other embodiments ofthe holding element include, for example, grasping elements such asmovable finger-like projections, and the like. The holding element isusually part of a transfer robot that comprises a robotic arm that iscapable or transferring the support from various positions where stepsin the synthesis of the chemical compound are performed such as betweenseveral flow devices in accordance with the present invention. In oneembodiment a transfer robot is mounted on the main platform. Thetransfer robot may comprise a base, an arm that is movably mounted onthe base, and an element for holding the support during transport thatis attached to the arm. Also included is a controller for controllingthe movement of the mechanism.

One embodiment of an apparatus in accordance with the present inventionis depicted in FIG. 3 in schematic form. Apparatus 200 comprisesplatform 201 on which the components of the apparatus are mounted.Apparatus 200 comprises main computer 202, with which various componentsof the apparatus are in communication. Video display 203 is incommunication with computer 202. Apparatus 200 further comprises printchamber 204, which is controlled by main computer 202. The nature ofprint chamber 204 depends on the nature of the printing techniqueemployed to add monomers to a growing polymer chain. Such printingtechniques include, by way of illustration and not limitation, inkjetprinting, and so forth. Camera 205 is in communication with maincomputer 202. Transfer robot 206 is also controlled by main computer 202and comprises a robot arm 208 that moves a support to be printed fromprint chamber 204 to first flow cell 210 or second flow cell 212 orstabilization flow cell 213. In one embodiment robot arm 208 introducesa support into print chamber 204 horizontally for printing on a surfaceof the support and introduces the support into a flow cell vertically.First flow cell 210 is in communication with program logic controller214, which is controlled by main computer 202, and second flow cell 212is in communication with program logic controller 216, which is alsocontrolled by main computer 202. First flow cell 210 is in communicationwith flow sensor and level indicator 218, which is controlled by maincomputer 202, and second flow cell 212 is in communication with flowsensor and level indicator 220, which is also controlled by maincomputer 202. First flow cell 210 is in fluid communication withmanifolds 222, 224 and 226, each of which is controlled by main computer202 and each of which is in fluid communication with a source of fluidreagents, namely, 234, 236 and 238, respectively. Second flow cell 212is in fluid communication with manifolds 228, 230 and 232, each of whichis controlled by main computer 202 and each of which is in fluidcommunication with a source of fluid reagents, namely, 240, 242 and 244,respectively. Stabilization flow cell 213 is in communication withprogram logic controller 215, which is controlled by main computer 202,and is in communication with flow sensor and level indicator 219, whichis controlled by main computer 202. Stabilization flow cell 213 is influid communication with manifolds 223 and 225, each of which iscontrolled by main computer 202 and each of which is in fluidcommunication with a source of fluid reagents, namely, 235 and 237,respectively.

The apparatus of the invention further comprise appropriate electricaland mechanical architecture and electrical connections, wiring anddevices such as timers, clocks, and so forth for operating the variouselements of the apparatus. Such architecture is familiar to thoseskilled in the art and will not be discussed in more detail herein.

The methods in accordance with the present invention may be carried outunder computer control, that is, with the aid of a computer. Forexample, an IBM® compatible personal computer (PC) may be utilized. Thecomputer is driven by software specific to the methods described herein.A preferred computer hardware capable of assisting in the operation ofthe methods in accordance with the present invention involves a systemwith at least the following specifications: Pentium® processor or betterwith a clock speed of at least 100 MHz, at least 32 megabytes of randomaccess memory (RAM) and at least 80 megabytes of virtual memory, runningunder either the Windows 95 or Windows NT 4.0 operating system (orsuccessor thereof).

Software that may be used to carry out the methods may be, for example,Microsoft Excel or Microsoft Access, suitably extended via user-writtenfunctions and templates, and linked when necessary to stand-aloneprograms that perform other functions. Examples of software or computerprograms used in assisting in conducting the present methods may bewritten, preferably, in Visual BASIC, FORTRAN and C⁺⁺. It should beunderstood that the above computer information and the software usedherein are by way of example and not limitation. The present methods maybe adapted to other computers and software. Other languages that may beused include, for example, PASCAL, PERL or assembly language.

A computer program may be utilized to carry out the above method steps.The computer program provides for (i) placing a support into a chamberfor printing a predetermined arrangement of features on the surface ofthe support, (ii) dispensing reagents for a specific cycle of chemicalreactions involved in the synthesis of compounds at the feature sites,(iii) activating a mechanism for determining the occurrence of an errorin the deposition of reagents, (iv) either (a) if an error is detected,moving the support to a stabilization chamber and subsequently movingthe support to a printing chamber to re-print the entire surface or, ifno error is detected, moving the support to a chamber for flooding ofthe support surface with a reagent involved in the synthesis of thechemical compounds or (b) identifying features on the support that werenot printed correctly and re-printing only those features, (v) removingthe support from the housing chamber, (vi) placing the support into achamber of a flow device, (vii) introducing a fluid reagent forconducting a reaction step into the reagent chamber, (viii) removing thefluid reagent from the reagent chamber, (ix) removing the support fromthe housing chamber and (x) moving the support to the printing chamberto conduct the next cycle in the synthesis of the chemical compound.

The computer program may be carried on a program product which includesa computer readable storage medium having a computer program storedthereon and which, when loaded into a programmable processor, providesinstructions to the processor of that apparatus such that it willexecute the procedures required of it to perform a method of the presentinvention. The computer readable storage medium may be an optical,magnetic, or solid state memory, any of which may be portable or fixed.

Another aspect of the present invention is a computer program productcomprising a computer readable storage medium having a computer programstored thereon which, when loaded into a computer, performs theaforementioned method.

Following receipt by a user of an array made by an apparatus or methodof the present invention, it will typically be exposed to a sample (forexample, a fluorescent-labeled polynucleotide or protein containingsample) and the array is then read. Reading of the array may beaccomplished by illuminating the array and reading the location andintensity of resulting fluorescence at each feature of the array. Forexample, a scanner may be used for this purpose where the scanner may besimilar to, for example, the AGILENT MICROARRAY SCANNER available fromAgilent Technologies Inc, Palo Alto, Calif. Other suitable apparatus andmethods are described in U.S. patent applications: Ser. No. 09/846,125“Reading Multi-Featured Arrays” by Dorsel, et al.; and Ser. No.09/430,214 “Interrogating Multi-Featured Arrays” by Dorsel, et al. Therelevant portions of these references are incorporated herein byreference. However, arrays may be read by methods or apparatus otherthan the foregoing, with other reading methods including other opticaltechniques (for example, detecting chemiluminescent orelectroluminescent labels) or electrical techniques (where each featureis provided with an electrode to detect hybridization at that feature ina manner disclosed in U.S. Pat. No. 6,221,583 and elsewhere). Resultsfrom the reading may be raw results (such as fluorescence intensityreadings for each feature in one or more color channels) or may beprocessed results such as obtained by rejecting a reading for a featurethat is below a predetermined threshold and/or forming conclusions basedon the pattern read from the array (such as whether or not a particulartarget sequence may have been present in the sample). The results of thereading (processed or not) may be forwarded (such as by communication)to a remote location if desired, and received there for further use(such as further processing).

When one item is indicated as being “remote” from another, this isreferenced that the two items are at least in different buildings, andmay be at least one mile, ten miles, or at least one hundred milesapart. “Communicating” information references transmitting the datarepresenting that information as electrical signals over a suitablecommunication channel (for example, a private or public network).“Forwarding” an item refers to any means of getting that item from onelocation to the next, whether by physically transporting that item orotherwise (where that is possible) and includes, at least in the case ofdata, physically transporting a medium carrying the data orcommunicating the data.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method for synthesizing an array of chemical compounds on thesurface of a support wherein said synthesis comprises a plurality ofsteps in which reagents for conducting said synthesis are deposited onthe surface of the support to form precursors of said chemical compoundsand wherein one or more of said plurality of steps may comprise an errorin said deposition, said method comprising: (a) depositing, on multiplelocations on said surface, reagents for forming said chemical compounds,(b) repeating step (a) in one or more cycles so as to form said chemicalcompounds, wherein the reagents deposited in different cycles at thesame locations on said surface may or may not be the same; the methodadditionally comprising in at least one selected cycle, (c) determiningwhether an error occurred in the depositing of said first reagents inthe selected cycle to one or more of said multiple locations, (d) if anerror occurred, (i) correcting the source of said error, and (ii)re-depositing on said surface at least some of those of said samereagents that were not correctly deposited in the selected cycle. 2-3.(canceled)
 4. A method for synthesizing a plurality of biopolymers onthe surface of a support wherein said synthesis comprises a plurality ofsteps in which reagents for conducting said synthesis are deposited onthe surface of the support to form precursors of said biopolymers andwherein one or more of said plurality of steps may comprise an error insaid deposition, said method comprising: (a) depositing on said surfacea reagent for forming said biopolymers, (b) repeating step (a) in one ormore cycles so as to form said biopolymers wherein the reagentsdeposited in different cycles at the same locations on said surface mayor may not be the same; the method additionally comprising in at leastone selected cycle, (c) determining using an imaging system, whether anerror occurred in the depositing of said reagent in the selected cycle,(d) if an error occurred, (i) treating said support to stabilizeprecursors of said biopolymers, (ii) correcting the source of saiderror, and (iii) re-depositing on said surface the same reagent of step(a) that was not correctly deposited in the selected cycle, and (e)preparing the support for the next depositing step.
 5. A methodaccording to claim 4 wherein said reagents are delivered to said surfaceas plurality of drops.
 6. A method according to claim 5 wherein saidreagents are delivered by means of a plurality of nozzles.
 7. A methodaccording to claim 5 wherein said error is determined by means ofcomparing an imprint of the delivered spots to a predetermined imprint.8. A method according to claim 7 wherein said imprint of said deliveredspots is determined by means of a camera.
 9. A method according to claim7 wherein said comparing is carried out by means of a computer. 10-11.(canceled)
 12. A method according to claim 4 wherein said treatingcomprises applying an oxidizing agent to said surface.
 13. A methodaccording to claim 12, which comprises washing said surface.
 14. Amethod for synthesizing an array of biopolymers on the surface of asupport wherein said synthesis comprises a plurality of steps whereinreagents comprising biopolymers subunits are deposited on the surface ofthe support and wherein one or more of said plurality of steps maycomprise an error in said deposition, said method comprising: (a)placing a support having a functionalized surface into a reactionchamber, (b) dispensing, to multiple locations on said surface from aplurality of nozzles, a plurality of drops of a reagent comprising abiopolymers subunit, (c) repeating steps (a) and (b) in one or morecycles so as to form said biopolymers, wherein the reagents deposited indifferent cycles at the same locations on said surface may or may not bethe same; the method additionally comprising in at least one selectedcycle, (d) determining, using an imaging system, whether an erroroccurred in the dispensing of said reagent in the selected cycle, (e) ifan error occurred, (i) removing said support from said reaction chamberand placing said support in a holding chamber, (ii) stabilizing thesurface of said support in said holding chamber, (iii) correcting thesource of said error, (iv) returning said support to said reactionchamber, and (v) dispensing, to said surface from a plurality ofnozzles, a plurality of drops of the same reagent for the cycle in whichthe error occurred and (f) subjecting said support to reagents forpreparing said support for a subsequent dispensing step.
 15. A methodaccording to claim 14 wherein said error is determined by means ofcomparing an imprint of the delivered spots to a predetermined imprint.16. A method according to claim 15 wherein said imprint of saiddelivered spots is determined by means of a camera.
 17. A methodaccording to claim 16 wherein said comparing is carried out by means ofa computer.
 18. A method according to claim 14 wherein said biopolymersare polynucleotides or polypeptides.
 19. A method according to claim 14wherein said stabilizing comprises applying an oxidizing agent to saidsurface.
 20. A method according to claim 19, which comprises washingsaid surface.
 21. A method according to claim 20 wherein said washing iscarried out in more than one step.
 22. A method according to claim 14for the synthesis of polynucleotides on said surface comprisingsubsequent to step (d) subjecting said surface to an oxidizing agent andsubjecting said surface to an agent for removing a protecting group. 23.(canceled)
 24. A method according to claim 14 further comprisingexposing the array to a sample and reading the array.
 25. A methodaccording to claim 24 comprising forwarding data representing a resultobtained from a reading of the array.
 26. A method according to claim 25wherein the data is transmitted to a remote location.
 27. A methodaccording to claim 24 comprising receiving data representing a result ofan interrogation obtained by the reading of the array.
 28. A methodaccording to claim 1 for synthesizing an array of biopolymers on thesurface of a support, said method utilizing an apparatus comprising: (a)a reaction chamber, (b) a mechanism for moving a support to and fromsaid reaction chamber, (c) a controller for controlling the movement ofsaid mechanism of (b), (d) one or more fluid dispensing stations influid communication with said reaction chamber, (e) a mechanism fordetermining the correct operation of said fluid dispensing stations,said mechanism being in communication with said controller forcontrolling the movement of said mechanism of (b), (f) a controller forcontrolling said mechanism of (e), and (g) a stabilization chamber forsubjecting said support to stabilization reagents. 29-30. (canceled) 31.A method according to claim 28 wherein said mechanism for determiningthe correct operation of said fluid dispensing stations is an inspectionsystem comprising a camera. 32-38. (canceled)